We have shown that FTD-specific CHMP2B mutant proteins cause enlarged late endosomes and disrupt endosome–lysosome fusion in cell culture models. Aberrant endosomes were also observed in patient brains and fibroblasts supporting the relevance of our in vitro overexpression model. We further dissect the function of the endocytic pathway to show that protein sorting is intact, indicating a potential pathogenic event downstream, at the level of fusion with lysosomes.
Cellular degradation of EGF, which is dependent on endosomal trafficking to the lysosome, was significantly decreased within the normal time course of this process. Furthermore, the amount of EGF reaching the lysosome was significantly reduced even after a 2 h chase. Western blotting analysis showed delayed degradation of the EGF receptor in response to EGF stimulation, this effect was marked over 2 h but less discernible at 3 h, suggesting that this process is impaired but not completely abrogated.
Sorting of EGFR onto the ILVs of MVBs is not functionally impaired, as shown by the normal time course of EGF-induced ERK1/2 activation and the presence of immunogold-labelled EGFR on the ILVs of abnormal endosomes in patient fibroblast cells. Knockdown of the ESCRT-III protein CHMP3 similarly prevents fusion of endosomes and lysosomes but not the deactivation of EGFR (15
). CHMP3 knockdown cells could sequester EGFR into endosomal structures, even though they formed fewer ILVs than control cells. Similarly, abnormal structures present in FTD-3 patient fibroblasts internalized EGFR despite some abnormal membrane deformation and sparsity of ILVs within the enlarged lumen. In CHMP5 knockout mice, ILV formation is also intact despite impairment of lysosomal degradation (16
). Taken together, this evidence suggests uncoupling of these two processes, and that some CHMP proteins may act downstream of ILV formation at the level of endosome–lysosome fusion. ILV formation, which includes deformation of the outer endosomal membrane and scission, is driven by the ESCRT-III complex (17
). In vitro
assays using the yeast homologues of the CHMP proteins, where one ESCRT-III subunit was omitted at a time, showed VPS2 (the homologue of CHMP2) to be unnecessary for both membrane deformation and scission (20
). Instead, Vps2 was shown to be necessary for recruiting the AAA+ ATPase VPS4, required for disassembly and recycling of ESCRT-III subunits.
The negatively charged C terminus of CHMP proteins functions as an auto-inhibitory domain, interacting with the positively charged N terminus to keep the protein in a ‘closed’ conformation which masks the membrane binding properties of the N terminus (21
). Deletion of the C terminus would therefore be predicted to produce a constitutively active protein which is likely to accumulate on endosomal membranes. Furthermore, the C terminus of CHMP2B is necessary for binding VPS4 (25
); and CHMP2B truncation mutants lose this VPS4 binding domain so would be predicted to fail to dissociate from the endosomal membrane. Dissociation-incompetent CHMP2B may allow ILVs to form normally but also sequester other ESCRT-III subunits at the endosomal membrane so that fewer ILVs can be formed over time.
We propose a model where the accumulation of constitutively bound CHMP2B on endosomes may render them incapable of fusion with lysosomes. This latter process is not well understood, but it may be necessary for ESCRT-III to dissociate before fusion occurs, and/or the constitutive presence of ESCRT-III may mean that the endosomal membrane is unable to recruit factors required for fusion. We demonstrate impaired recruitment of Rab7 onto endosomes in CHMP2B mutant cells. This may be relevant to the neurodegenerative process as mutations in RAB7
cause the motor and sensory neuropathy Charcot–Marie–Tooth type 2B (27
). Rab7 is a GTPase marking fusion-competent MVBs, which is recruited by the HOPS (ho
motypic vacuole fusion and vacuole p
orting) and CORVET (class C cor
ethering) complexes (14
). The proportion of endosomes that recruit Rab7 is reduced by approximately one-third in CHMP2B mutant cells but not completely diminished. This may explain why fusion is decreased and degradation delayed instead of completely abrogated. Interestingly, overexpression of EGFP-Rab7 did not rescue this deficit; the reduction in recruitment was the same for EGFP-Rab7 and endogenous Rab7. This is consistent with a model where mutant CHMP2B prevents a proportion of late endosomes from recruiting Rab7 and a greater concentration of Rab7 is therefore redundant to these recruitment-incompetent endosomes.
That cortical neurons are selectively vulnerable in the time course of disease remains a conundrum; although it may suggest that these cells are particularly dependent on some aspect of endosomal function for their survival. Normal trafficking of growth factors and their receptors is one possible candidate. Here we showed that epidermal growth factor remained undegraded over the normal time course within CHMP2B-positive enlarged compartments which failed to fuse with lysosomes. However, owing to its rapid sorting into the ILVs of MVBs, there was no prolongation of EGF signalling. Whereas EGF is rapidly internalized and activates its downstream signalling pathways over a short time course (30
), other neuronal growth factor-receptor complexes, such as BDNF-TrkB, are known to signal from the endosomal membrane over a longer period (31
). Therefore, further studies examining how these signalling endosomes are affected in neuronal models of CHMP2B mutations will be of great interest. The ESCRT complex is also involved in the endosomal–lysosomal degradation of neurotransmitter receptors (34
), so impairment of receptor homeostasis and excitotoxic insult is another potential neurotoxic mechanism that could be mediated by mutant CHMP2B proteins. Finally, studies in Drosophila
have recently shown that mutant CHMP2B can activate the Toll-like receptor pathway (36
): a process which has been implicated in neurodegeneration (37
In the present study, we have focused on endosomal dysfunction as the human pathology data so far support a major role for this system in the pathogenesis of FTD-3. It is of great interest that the endosome–lysosome pathway described here also converges with the autophagosomal degradation pathway (38
) and that other cellular studies of CHMP2B overexpression have shown a deficit in the fusion of autophagosomes with lysosomes (41
). Basal autophagy is required in neuronal cells to degrade organelles and long lived proteins which might otherwise accumulate with deleterious effects for the cell (44
). It is likely that both of these highly related processes contribute to the pathogenesis of FTD-3. It is thought that similar mechanisms mediate autophagosome–lysosome and endosome–lysosome fusion (reviewed in 29
), so data presented here for a putative role for impaired Rab7 recruitment may also be of relevance to the reported deficits in autophagy.
The findings presented here strongly support a model where C-terminal truncations of CHMP2B are pathogenic by a toxic gain-of-function which disrupts endocytic trafficking, producing deleterious effects for neuronal cells. Defects in endosomal trafficking have been implicated in a number of neurodegenerative diseases and the novel phenotypic and functional assays described here may aid diagnosis and the eventual screening and development of therapeutics.